LOCUS       Exported                7189 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  An AAV vector expressing S. aureus dCas9 fused to VP64..
ACCESSION   .
VERSION     .
KEYWORDS    pCMV-sadCas9-VP64
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7189)
  AUTHORS   Matharu N, Rattanasopha S, Tamura S, Maliskova L, Wang Y, Bernard A,
            Hardin A, Eckalbar WL, Vaisse C, Ahituv N
  TITLE     CRISPR-mediated activation of a promoter or enhancer rescues obesity
            caused by haploinsufficiency.
  JOURNAL   Science. 2018 Dec 13. pii: science.aau0629. doi: 
            10.1126/science.aau0629.
  PUBMED    30545847
REFERENCE   2  (bases 1 to 7189)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7189
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     133..152
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(371..475)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     543..561
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(599..621)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     721..740
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(757..1212)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(894..915)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1106..1125
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   1287..1427
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   1298..1427
                     /label=AAV2 ITR
     enhancer        1447..1826
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1827..2030
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1981..2001
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      2049..2058
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             2061..2081
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5262..5309
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5340..5489
                     /codon_start=1
                     /product="tetrameric repeat of the minimal activation 
                     domain of herpes simplex virus VP16 (Beerli et al., 1998)"
                     /label=VP64
                     /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF
                     DLDML"
     primer_bind     complement(5523..5540)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5529..5736
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5749..5878
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     rep_origin      complement(5940..6528)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6020..6039)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6699..370)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
ORIGIN
        1 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
       61 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
      121 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
      181 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
      241 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
      301 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
      361 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
      421 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
      481 ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata
      541 aaaataggcg tatcacgagg ccctttcgtc tcgcgcgttt cggtgatgac ggtgaaaacc
      601 tctgacacat gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca
      661 gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg tcggggctgg cttaactatg
      721 cggcatcaga gcagattgta ctgagagtgc accataaaat tgtaaacgtt aatattttgt
      781 taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag accgaaatcg
      841 gcaaaatccc ttataaatca aaagaatagc ccgagataga gttgagtgtt gttccagttt
      901 ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct
      961 atcagggcga tggcccacta cgtgaaccat cacccaaatc aagttttttg gggtcgaggt
     1021 gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa
     1081 agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctaaggcgc
     1141 tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc
     1201 tacagggcgc gtactatggt tgctttgacg tatgcggtgt gaaataccgc acagatgcgt
     1261 aaggagaaaa taccgcatca ggcgcccctg caggcagctg cgcgctcgct cgctcactga
     1321 ggccgcccgg gcaaagcccg ggcgtcgggc gacctttggt cgcccggcct cagtgagcga
     1381 gcgagcgcgc agagagggag tggccaactc catcactagg ggttcctgcg gccgcctcga
     1441 ggcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc
     1501 atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac
     1561 cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa
     1621 tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag
     1681 tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc
     1741 ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct
     1801 acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg
     1861 gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt
     1921 tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga
     1981 cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct ctctggctaa
     2041 ctaccggtgc caccatggcc ccaaagaaga agcggaaggt cggtatccac ggagtcccag
     2101 cagccaagcg gaactacatc ctgggcctgg ccatcggcat caccagcgtg ggctacggca
     2161 tcatcgacta cgagacacgg gacgtgatcg atgccggcgt gcggctgttc aaagaggcca
     2221 acgtggaaaa caacgagggc aggcggagca agagaggcgc cagaaggctg aagcggcgga
     2281 ggcggcatag aatccagaga gtgaagaagc tgctgttcga ctacaacctg ctgaccgacc
     2341 acagcgagct gagcggcatc aacccctacg aggccagagt gaagggcctg agccagaagc
     2401 tgagcgagga agagttctct gccgccctgc tgcacctggc caagagaaga ggcgtgcaca
     2461 acgtgaacga ggtggaagag gacaccggca acgagctgtc caccaaagag cagatcagcc
     2521 ggaacagcaa ggccctggaa gagaaatacg tggccgaact gcagctggaa cggctgaaga
     2581 aagacggcga agtgcggggc agcatcaaca gattcaagac cagcgactac gtgaaagaag
     2641 ccaaacagct gctgaaggtg cagaaggcct accaccagct ggaccagagc ttcatcgaca
     2701 cctacatcga cctgctggaa acccggcgga cctactatga gggacctggc gagggcagcc
     2761 ccttcggctg gaaggacatc aaagaatggt acgagatgct gatgggccac tgcacctact
     2821 tccccgagga actgcggagc gtgaagtacg cctacaacgc cgacctgtac aacgccctga
     2881 acgacctgaa caatctcgtg atcaccaggg acgagaacga gaagctggaa tattacgaga
     2941 agttccagat catcgagaac gtgttcaagc agaagaagaa gcccaccctg aagcagatcg
     3001 ccaaagaaat cctcgtgaac gaagaggata ttaagggcta cagagtgacc agcaccggca
     3061 agcccgagtt caccaacctg aaggtgtacc acgacatcaa ggacattacc gcccggaaag
     3121 agattattga gaacgccgag ctgctggatc agattgccaa gatcctgacc atctaccaga
     3181 gcagcgagga catccaggaa gaactgacca atctgaactc cgagctgacc caggaagaga
     3241 tcgagcagat ctctaatctg aagggctata ccggcaccca caacctgagc ctgaaggcca
     3301 tcaacctgat cctggacgag ctgtggcaca ccaacgacaa ccagatcgct atcttcaacc
     3361 ggctgaagct ggtgcccaag aaggtggacc tgtcccagca gaaagagatc cccaccaccc
     3421 tggtggacga cttcatcctg agccccgtcg tgaagagaag cttcatccag agcatcaaag
     3481 tgatcaacgc catcatcaag aagtacggcc tgcccaacga catcattatc gagctggccc
     3541 gcgagaagaa ctccaaggac gcccagaaaa tgatcaacga gatgcagaag cggaaccggc
     3601 agaccaacga gcggatcgag gaaatcatcc ggaccaccgg caaagagaac gccaagtacc
     3661 tgatcgagaa gatcaagctg cacgacatgc aggaaggcaa gtgcctgtac agcctggaag
     3721 ccatccctct ggaagatctg ctgaacaacc ccttcaacta tgaggtggac cacatcatcc
     3781 ccagaagcgt gtccttcgac aacagcttca acaacaaggt gctcgtgaag caggaagaag
     3841 ccagcaagaa gggcaaccgg accccattcc agtacctgag cagcagcgac agcaagatca
     3901 gctacgaaac cttcaagaag cacatcctga atctggccaa gggcaagggc agaatcagca
     3961 agaccaagaa agagtatctg ctggaagaac gggacatcaa caggttctcc gtgcagaaag
     4021 acttcatcaa ccggaacctg gtggatacca gatacgccac cagaggcctg atgaacctgc
     4081 tgcggagcta cttcagagtg aacaacctgg acgtgaaagt gaagtccatc aatggcggct
     4141 tcaccagctt tctgcggcgg aagtggaagt ttaagaaaga gcggaacaag gggtacaagc
     4201 accacgccga ggacgccctg atcattgcca acgccgattt catcttcaaa gagtggaaga
     4261 aactggacaa ggccaaaaaa gtgatggaaa accagatgtt cgaggaaaag caggccgaga
     4321 gcatgcccga gatcgaaacc gagcaggagt acaaagagat cttcatcacc ccccaccaga
     4381 tcaagcacat taaggacttc aaggactaca agtacagcca ccgggtggac aagaagccta
     4441 atagagagct gattaacgac accctgtact ccacccggaa ggacgacaag ggcaacaccc
     4501 tgatcgtgaa caatctgaac ggcctgtacg acaaggacaa tgacaagctg aaaaagctga
     4561 tcaacaagag ccccgaaaag ctgctgatgt accaccacga cccccagacc taccagaaac
     4621 tgaagctgat tatggaacag tacggcgacg agaagaatcc cctgtacaag tactacgagg
     4681 aaaccgggaa ctacctgacc aagtactcca aaaaggacaa cggccccgtg atcaagaaga
     4741 ttaagtatta cggcaacaaa ctgaacgccc atctggacat caccgacgac taccccaaca
     4801 gcagaaacaa ggtcgtgaag ctgtccctga agccctacag attcgacgtg tacctggaca
     4861 atggcgtgta caagttcgtg accgtgaaga atctggatgt gatcaaaaaa gaaaactact
     4921 acgaagtgaa tagcaagtgc tatgaggaag ctaagaagct gaagaagatc agcaaccagg
     4981 ccgagtttat cgcctccttc tacaacaacg atctgatcaa gatcaacggc gagctgtata
     5041 gagtgatcgg cgtgaacaac gacctgctga accggatcga agtgaacatg atcgacatca
     5101 cctaccgcga gtacctggaa aacatgaacg acaagaggcc ccccaggatc attaagacaa
     5161 tcgcctccaa gacccagagc attaagaagt acagcacaga cattctgggc aacctgtatg
     5221 aagtgaaatc taagaagcac cctcagatca tcaaaaaggg caaaaggccg gcggccacga
     5281 aaaaggccgg ccaggcaaaa aagaaaaagg gatccgaggc cagcggttcc ggacgggctg
     5341 acgcattgga cgattttgat ctggatatgc tgggaagtga cgccctcgat gattttgacc
     5401 ttgacatgct tggttcggat gcccttgatg actttgacct cgacatgctc ggcagtgacg
     5461 cccttgatga tttcgacctg gacatgctgt gatctagaga attcctagag ctcgctgatc
     5521 agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc
     5581 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc
     5641 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg
     5701 ggaggattgg gaagagaata gcaggcatgc tggggagcta gaggccgcag gaacccctag
     5761 tgatggagtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa
     5821 aggtcgcccg acgcccgggc ggcctcagtg agcgagcgag cgcgcagctg cctgcaggac
     5881 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     5941 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     6001 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     6061 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     6121 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     6181 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     6241 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     6301 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     6361 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     6421 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     6481 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     6541 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     6601 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     6661 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     6721 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     6781 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcgg
     6841 cttccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     6901 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     6961 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     7021 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     7081 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     7141 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatgg
//
